Various devices and methods for determining the optical density of reaction mixtures in shaken bioreactors are known in the art. The generally known principle behind these technologies is the diffusion and/or transmission of light through matter located on the light path. The diffusion and transmission intensities are in a relationship, which can be modelled mathematically, with the concentration of the matter located on the light path which interacts with the light, and so measuring the intensities of scattered and transmitted light makes conclusions as to substance concentrations possible. Devices for implementing this basic method conventionally consist of at least one light source for introducing light into the volume to be analysed and at least one light sensor for detecting the scattered and/or transmitted light. This underlying construction and the underlying method can be modified to achieve better results. The prior art publications cited hereinafter are therefore only described in so far as they go beyond and improve the generally known methods and devices for determining the optical density of reaction mixtures.
US 2009/0075248 A1 discloses a method and a device for optically determining the particle concentration in a medium over a wide linear range. In this document, a plurality of light source/light sensor pairs are used for measuring scattering and/or transmission signals, which can in turn be combined using various algorithms in such a way that there is a linear dependency between the algorithmically modified measurement signal and the particle concentration in the medium over a range of up to three orders of magnitude of concentration (for example 0.1 g/l to 100 g/l). According to the patent specification, this simplifies the optical determination of the particle concentration in a medium, since the normally non-linear relationship between the particle concentration and a single directly measured signal is transformed into a linear relationship. Various embodiments of the method and device are disclosed, for example for non-invasive measurement of the biomass concentration in a fermenter through the translucent wall thereof, for non-invasive measurement of the biomass concentration in unshaken flasks through the light-permeable walls thereof, or for invasive measurement by means of a measurement probe which can be immersed in the medium.
The primary drawback of the method and device of US 2009/0075248 A1 is that it cannot be used for measurement in shaken systems during shaking operation. The constantly changing shape and distribution of the medium in the shaken vessel during shaking lead to periodically fluctuating strengths of the transmission and scattering signal, which are not taken into account by the disclosed method and device and thus render the method unusable. A further drawback of the disclosed embodiments is that they cannot be applied to a shaken reactor, or can only be applied with poor mechanical stability.
In addition, as regards the method and device of US 2009/0075248 A1, it is doubtful whether the modes of operation, measurement ranges and measurement precisions set out in the patent specification are actually achievable in a real-life application, in particular in shaken systems. The examples set out in the patent specification are only of a limited predictive power, at least for applications in the field of cultivating organisms, since they are carried out on yeast suspensions in aqueous 0.9% NaCl solution. However, the culture solutions used in reality have to contain organic nutrition sources (for example protein lysates, cell extracts, sugars, amino acids, lipids etc.), which also contribute to the strength of the scattering and transmission signal to be measured. This leads to different behaviour of the disclosed linearization algorithms when the same cell concentration is measured in different media, since these algorithms are dependent on signal thresholds, and whether these thresholds are reached is affected to different degrees by different media. The resulting measurement imprecisions are a major drawback of the method and device.
U.S. Pat. No. 8,405,033 B2 discloses a method and a device for the optical determination of the particle concentration in a medium through the side wall of a container. The particle concentration is determined exclusively by way of the scattered light; no transmission signal is measured. So as to be able to work with low liquid levels, a wavelength is used which is strongly absorbed by the medium, in such a way that the measured scattering signal originates merely from a small volume directly in front of the sensor and light source, which are positioned at a distance from one another of at least 10% and at most 1000% of the average penetration depth of the light into the absorbing medium. Various configurations of the method and device are disclosed, for example for non-invasive measurement of the biomass concentration through the side wall or the base of a shaking flask. Further, one sentence mentions recording several measurement points per second so as to observe fluctuations in the liquid level in front of the detector. However, it is not disclosed what measurement purpose this is intended to serve.
A drawback of the method and device of U.S. Pat. No. 8,405,033 B2 is the limitation to measurements on the scattering signal, even though at low particle concentrations (for example cell densities having OD600<0.5) the precision and reliability of transmission measurements are much better than for scattering measurements.
A further drawback of the method and device of U.S. Pat. No. 8,405,033 B2 is the practicability thereof in real appliances. Thus, for correct operation of the “OD scanner” appliance marketed by BugLab LLC, 3350 Clayton Road, Suite 220, Concord, Calif. 94519 on the basis of this patent, a liquid level of at least 3 cm is required in front of the light source and the detector. Specifically when small reaction vessels (for example shaking flasks) are used, this can only be achieved by way of oblique positioning, meaning that the measurement cannot be taken during shaking operation. Measurements by way of a system attached below the flask as proposed in the patent also cannot be implemented in practice for the small volumes of media and reaction vessels frequently used in the industry (for example 20 ml in a 200 ml shaking flask), since the required fill levels cannot be reached for conventional flask fill amounts of 10%. Therefore, so as to be able to take continuous measurements during shaking operation on the basis of this patent, measurements either in very large medium volumes (for example 200 ml in a 2000 ml shaking flask) or in considerably overfilled shaking flasks (fill amount>>10%) would be required. Neither of these variants makes any sense, since overfilled shaking flasks have very poor mixing and oxygen transfer rates which cannot be scaled to other reactor systems, and large-volume shaking flask experiments go against the principle of minimised high-throughput screening.
In addition, as regards the method and device of U.S. Pat. No. 8,405,033 B2, as was the case for US 2009/0075248 A1, it is doubtful whether the modes of operation, measurement ranges and measurement precisions set out in the patent specification are actually achievable in a real-life application, in particular in shaken systems. The examples set out in the patent specification were also carried out on yeast suspensions in aqueous 0.9% NaCl solution, and are only of limited predictive power, as per the arguments regarding US 2009/0075248 A1, at least for applications in the field of cultivating organisms. In particular in the aforementioned measurement range of low particle concentrations, the scattered light measurement can be distorted by typically optically active substances in the medium. The calibration of the measurement appliance to each newly used medium, as required to correct the error, is therefore an additional drawback.
JP 02/012217426 A discloses a method for contactless, continuous measurement of the growth of a sample during cultivation. The basic functional principle of the method is transmitted and scattered light measurement at a position where there is only a very low liquid thickness as a result of the liquid distribution occurring during shaking, in such a way that evaluable transmission measurements can be carried out. Further, the possibility of measurement value correction using various parameters is disclosed. Automated addition and removal of culture media with feedback to the measurement values is also mentioned. Adaptation of the light source and detector position with feedback to the measurement values for the best possible optimisation of the measurement is also disclosed.
A drawback of the method of JP 02/012217426 A is the low width of the measurement range (maximum OD600=20) derived from the patent, meaning that measurements cannot be taken at higher cell densities. In the low concentration range (OD600<1), the precision of the method is poor, as can be seen from the examples set out in the patent specification. These drawbacks are also apparent from the product manufactured by the patent proprietor, TAITEC Corp., 2693-1, Nishikata, Koshigaya City, Saitama, Japan, the measurement range of which, at OD600 values between 0.1 and 2.0, is much narrower and thus of less use than for the aforementioned prior art patents.
A further major drawback of JP 02/012217426 A is the strong dependency of the measurement on the shaking frequency (JP 02/012217426 A, Table 2). To apply the method at different shaking frequencies, specific correction or calibration data are therefore required in each case, and recording these constitutes a drawback in application in practice.
A further drawback of the method of JP 02/012217426 A is the susceptibility thereof to ambient light. The measurement on particularly thin liquid layers using a light source and a detector can lead to distortion of the measurement due to ambient light sources. The patent does mention that the use of infrared light can eliminate this source of error; however, conventional light bulbs and the heated walls of temperature-controlled incubators both generate non-negligible amounts of infrared radiation, and can thus also contribute to measurement errors.
An additional drawback of the method of JP 02/012217426 A is the manner in which the light sensors and light sources are arranged, as disclosed in the embodiment and implemented in the corresponding product (“OD-Monitor”) from TAITEC Corp., 2693-1, Nishikata, Koshigaya City, Saitama, Japan. In particular but not exclusively in relation to measurement on thin liquid levels as disclosed in the patent, measurement in parallel with the shaking plane has the major drawback that specific shaking frequencies, reactor shapes and fill levels are required so as to bring the medium to be analysed into the optical path between the light source and the light sensor. This disadvantageously limited the universal use of the device for different reactor sizes, shapes and fill levels. In addition, changes, due to the process, in the physical and fluid-dynamic parameters of the shaken medium lead to the thickness of the liquid level located on the light path changing, and this can lead to significant measurement errors or even to complete immeasurability (in the complete absence of a liquid film in the optical path). Examples of changes of this type in the medium are the change in viscosity due to the increase in biomass, due to filamentous growth or due to the secretion of gel-forming substances and the change in the medium volume due to evaporation effects.
U.S. Pat. Nos. 6,673,532 B2 and 7,041,493 B2 disclose a method and a device for the optochemical observation of bioprocesses. Among other things, very generally the possibility of determining the optical density of the culture broth by way of transmission measurements is also disclosed. Likewise, the possibility of a process carried out while shaking and the addition of fluids into the culture broth with feedback to measured parameters are mentioned.
A drawback of the method and device of U.S. Pat. Nos. 6,673,532 B2 and 7,041,493 B2 is the invasive nature of the optical-fibre-based measurement of the optical density, since it results in a high risk of contaminating the culture broth. In addition, this method of measuring optical densities can only be implemented with great difficulty and with mechanical instability in reactors having a shape other than an ideal cylinder shape (for example shaking flasks). The lack of scattered-light-based measurements of the optical density is also a major drawback, since transmission measurements by the method set out therein have a high susceptibility to errors and measurement imprecision at higher biomass concentrations (for example OD600>3) as a result of the very low transmissivity in these cases. At even higher biomass concentrations, such as occur for example in high-cell-density fermentations, the transmissivity falls towards zero, in such a way that evaluable measurement results can no longer be achieved in these cases without scattered light analyses.
A further drawback of the method and device of U.S. Pat. Nos. 6,673,532 B2 and 7,041,493 B2 is the need to use a shaker and positioning table specifically matched to the optical devices, since this means that pre-existing shaking appliances cannot be expanded with an analysis device. The combination set out in the patents of a positioning table having a dispenser for adding fluids to the culture broth is also disadvantageous, since the use of one dispenser for a plurality of reactors brings the risk of cross-contamination. Thus, during the dispensing process, droplets and aerosols from a reactor which are produced by the shaking movement can come into contact with the dispenser, which subsequently passes them on to another reactor during the next dispensing process, thus potentially contaminating it with foreign cells and biomolecules or toxic medium constituents.
DE 10 2004 017039 A1 discloses a method and a device for detecting process parameters of reaction liquids in a plurality of shaken microreactors. The determination of the biomass concentration by way of scattered light measurements is also part of the description.
A drawback of the method and device of DE 10 2004 017039 A1 is the lack of transmission measurements, leading to a worse precision of measurements at low cell densities (for example OD600<0.5) as stated previously. A further drawback is the limitation of the method and device to microreactors, even though many biotechnological, biochemical, microbiological, pharmaceutical and chemical screening processes are carried out at volumes>1 ml.
A further drawback of the method and device of DE 10 2004 017039 A1 is the idea of measurement on a shaken reactor using a stationary, unshaken light source/detector combination. The pulsed illumination of the reactor tuned to the shaking movement, as required for this method and disclosed in the patent, does guarantee that the light is always incident at the same point on the reactor. However, this method can only provide correct measurement data if the same part of the liquid distribution due to shaking is also always located at the measurement position at the moment of each measurement. However, as a result of the aforementioned changes in liquid volume and viscosity which accompany the process, the liquid distribution due to shaking in the reactor may change in such a way that the corresponding relative measurement position within this liquid distribution also changes, in such a way that the measurement results are no longer comparable with the results recorded earlier in the process, and correct determination of the optical density is no longer possible with the occurring fluid-mechanical changes.
All methods and devices known in the art for determining the optical density and/or the change in the optical density of reaction mixtures in shaken reactors are based on an underlying measurement approach which attempts to use particular measures to minimise or eliminate the effect on a measurement from the movement of the reaction mixture in the reactor due to shaking. The measures used for this purpose are for example interrupting the shaking (U.S. Pat. No. 8,405,033 B2), selecting a light path on which the movement of the reaction mixture due to shaking is minimal (JP 02/012217426A), immersing optical fibres to optimise the light path (U.S. Pat. Nos. 6,673,532 B2 and 7,041,493 B2) and using flash lamps tuned to the shaking frequency as a light source (DE 10 2004 017039 A1).